Uropathogenic Escherichia coli (UPEC), the primary cause of urinary tract infections (UTIs), have evolved exquisite mechanisms to subvert host defenses and to persist within the urinary tract in both intra- and extracellular niches. Within th intracellular site, UPEC undergo a developmental process that parallels extracellular biofilm formation and culminates in the biogenesis of intracellular bacterial communities (IBCs) that contain tightly packed bacteria encased within an extracellular matrix. Thus, identification of determinants that control bacterial intracellular replication will advance our understanding of UTI pathogenesis, will delineate strategies utilized by pathogens to occupy intracellular sites during infection, and will elucidate novel targets for anti-virulence therapy. Deletion of a UPEC factor, Colicin V production Accessory protein (CvpA), abolishes extracellular biofilm formation without influencing production of type 1 pili. Aiming to understand the contribution of CvpA to UPEC pathogenesis, I tested a UPEC cvpA deletion mutant in a murine model of acute UTI and determined that loss of CvpA abrogated the ability of UPEC to form IBCs during acute bladder infection despite WT levels of adherence to and subsequent invasion of superficial bladder epithelial cells in vivo. I also discovered that CvpA-deficient mutants are more susceptible to killing by neutrophils. The only described function for CvpA is that in its absence, colicin V, a small pore-forming toxin is not detected in the extracellular milieu. Although the plasmids that encode the colicin V structural and transport genes are not harbored by all UPEC strains (including strain UTI89, used in these studies), CvpA is conserved among E. coli, suggesting that CvpA has additional functions possibly such as the production and/or secretion of other pore- forming toxins. I hypothesize that during infection (1) CvpA modulates UPEC-host cell interactions and may be involved in a) dampening the host immune response and/or b) egressing from the vacuole upon entry into the host cells; and (2) loss of CvpA function impairs UPEC defenses against anti-bacterial assaults. In Aim 1 of this proposal, I will determine how loss of CvpA attenuates UPEC virulence. These studies will employ murine models of acute urinary tract infection, immunological assays, and advanced whole organ imaging methodologies. In Aim 2, I will interrogate the effects of cvpA deletion on UPEC toxin- associated factors and membrane integrity, using classical genetic, biochemical, and mass spectrometry approaches, and susceptibility assays. In total, these studies will delineate how CvpA promotes colonization of the urinary tract, will identify the mechanism by which CvpA modulates pathogenesis, and will evaluate CvpA as a bacterial target for drug development.